In vitro flow cytometry assay to assess primary human and mouse macrophage phagocytosis of live cells

Summary Although tumor-associated macrophages are generally immunosuppressive, macrophages may also promote tumor clearance via phagocytosis of live tumor cells. Here, we present a protocol for assessing macrophage engulfment of tumor cells in vitro using flow cytometry. We describe steps for cell preparation, reseeding macrophages, and setting up phagocytosis. We then detail procedures for collecting samples, staining macrophages, and flow cytometry. The protocol is applicable to both mouse bone-marrow-derived macrophages and human monocyte-derived macrophages. For complete details on the use and execution of this protocol, please refer to Roehle et al. (2021).1


SUMMARY
Although tumor-associated macrophages are generally immunosuppressive, macrophages may also promote tumor clearance via phagocytosis of live tumor cells. Here, we present a protocol for assessing macrophage engulfment of tumor cells in vitro using flow cytometry. We describe steps for cell preparation, reseeding macrophages, and setting up phagocytosis. We then detail procedures for collecting samples, staining macrophages, and flow cytometry. The protocol is applicable to both mouse bone-marrow-derived macrophages and human monocyte-derived macrophages. For complete details on the use and execution of this protocol, please refer to Roehle et al. (2021). 1

BEFORE YOU BEGIN
The protocol below describes the specific steps for assessing primary human and mouse macrophage phagocytosis of live cells.

Institutional permissions
These studies were conducted in accordance with the Declaration of Helsinki and the Belmont Report. Anonymous healthy donor leukopacks were obtained from the Kraft Family Blood Donor Center at Dana-Farber Cancer Institute and Brigham and Women's Hospital, protocol T0363. Institutional permission must be obtained prior to working with human blood.
Mice were housed at the Dana-Farber Cancer Institute and were maintained according to protocols approved by the DFCI Institutional Animal Care and Use Committee (IACUC) (#14-019 and #14-037). Institutional permission must be obtained prior to working with live mice.

Preparation of macrophages
Timing: 5 days 1. Differentiation of mouse macrophages: a. Isolate mouse bone marrow. i. Euthanize mouse by CO 2 asphyxiation or other approved method as specified by institutional guidelines for humane euthanasia. ii. Spray mouse abdomen and hind legs with 70% ethanol. iii. Open the abdomen using sterile scissors and remove the skin from each hind leg. iv. Cut each hind leg above the hip joint and below the knee joint to isolate the femur with adjacent joints intact. Be careful not to break the femurs. v. Using scissors, forceps, and Kimwipes, gently clean off the muscles and tissues from the bones. vi. Rinse the bones once in 70% ethanol and then twice in PBS. vii. Once all bones are collected and rinsed, move to a sterile biosafety cabinet. viii. Prepare a 10-mL syringe with a 27-gauge needle and fill the syringe with ice-cold PBS. ix. Cut off the ends of each bone using sterile scissors. Using the syringe, flush out the bone marrow into a sterile dish. Continue flushing until the bone turns translucent, using more PBS as needed. x. Once all bone marrow is collected, gently pipet the mixture up and down with a 10 mL serological pipette, then filter the mixture through a 40 mM filter into a 50 mL conical tube. xi. Centrifuge at 400 3 g for 5 min at 4 C to pellet cells and aspirate the supernatant. b. Lyse red blood cells. i

OPEN ACCESS
iii. Plate 1-2 million cells per mL onto petri dishes and incubate at 37 C and 5% CO 2 for five days. d. Feed cells on day 2 and day 4 of culture.
i. For each petri dish, add the same volume of RPMI complete with 50 ng/mL of M-CSF as originally plated. If the plate is too full, aspirate half of the previous media. Otherwise, add fresh media to the existing volume.

Maintenance of target tumor cells
Timing: 5 days 3. Culture target tumor cells: a. Thaw a cryogenic vial of the tumor cells briefly in a 37 C water bath. b. Mix the thawed cells with 9 mL of pre-warmed (37 C) RPMI complete in a 15 mL conical tube. c. Centrifuge at 400 3 g for 5 min at 4 C. d. Resuspend cells in 10 mL of pre-warmed (37 C) RPMI complete and transfer to a 75cm 2 tissue culture-treated flask. e. Incubate at 37 C and 5% CO 2 .
CRITICAL: The tumor cells will need to be able to grow in RPMI complete with M-CSF in order to be cultured with the macrophages during the assays. Some cell lines may do better with being slowly adjusted to RPMI complete in culture before the assay. Both adherent and non-adherent cells are compatible with the protocol. Non-adherent cells should be centrifuged to the bottom of the plate to facilitate rapid contact with macrophages. Steps for setting up phagocytosis assay

Timing: 1 day
On the day of the assay, tumor cells are washed and dissociated from their flask and plated onto macrophages. If the tumor cells are not fluorescent, then additional steps are needed to label the tumor cells before plating. Different treatments may be added prior to or during macrophage and tumor cell coculture to assess their impact on phagocytosis. Here, we include the SMAC mimetic LCL-161 and cytokines IFN-g or lymphotoxin as agents known to induce phagocytosis of live tumor cells. 1,2 The tumor cells should be as healthy as possible before being plated onto the macrophages, so keep the cells on ice during any waiting periods and try to move quickly.  Optional: If the macrophages are pretreated, wash the 12-well plates three times with PBS before adding tumor cells. In order to avoid detachment of the macrophages, tilt the plate at a 45 angle to gently aspirate the supernatant from the side of the well and slowly pipet 1 mL of PBS onto the side of each well.

Collect tumor cells (*optional for non-adherent cells, collect cells and start
7. Plate tumor cells onto the macrophages. a. Prepare three replicate wells for each condition tested. b. If there is no treatment in the coculture, add 1 mL of the 13 tumor cell solution into each well of macrophages. c. If there is treatment in the coculture, first add 500 mL of the 23 tumor cell solution and then add 500 mL of the 23 treatment solution into each well. d. Remember to include a macrophage-only well and a tumor cell-only well as negative controls to help set the flow cytometry gates. e. After plating, gently rock the plate from front to back and side to side to distribute the cells evenly. 8. Incubate at 37 C with 5% CO 2 for 18-24 h.

Timing: 2 h
After 18-24 h of coculture, the cells are ready to be collected, stained, and fixed for flow cytometry. The cells are dissociated from each well, collected into flow tubes, and stained for CD45 to identify the macrophages. The samples are then fixed and can be processed on a flow cytometer. All of the following steps can be performed on the benchtop. 9. Prepare flow cytometry tubes with 2 mL of HBSS for each well of the assay. 10. Pipet the 1 mL of supernatant from each well into the corresponding tube. This collects any nonadherent cells. 11. Add 1 mL of trypsin 0.25% EDTA into each well. 12. Incubate plates for 15 min at 37 C. 13. Collect the trypsinized cells into the corresponding flow tubes.
a. The plates and tubes should be kept on ice during this process. b. Hold the plate at a 45 angle. Try to aim the pipette at different spots around the circumference of the well while pipetting up and down. The same tip can be used for wells of the same condition (troubleshooting 1). c. Check the plates under the microscope to ensure full collection of cells. 14. Centrifuge at 400 3 g for 5 min at 4 C.  Figure 1). Here, we confirm that culturing macrophages with LCL-161 and IFN-g or lymphotoxin induces phagocytosis of tumor cells 1 (Figure 2). Although both cytokines can combine with LCL-161 to induce phagocytosis, lymphotoxin is better at inducing phagocytosis in mouse macrophages whereas IFN-g is better at inducing phagocytosis in human macrophages ( Figure 3).

LIMITATIONS
While this assay gives a quantitative measure of tumor cell uptake by macrophages, it is not possible to distinguish whether the macrophages are engulfing live tumor cells or if they are taking up remnants from dead cells. 3 We used video microscopy to show that LCL and lymphotoxin-treated macrophages do engulf live tumor cells, 1 but this cannot be directly concluded from a flow-cytometry based phagocytosis assay. Therefore, it is important to keep the tumor cells healthy when plating onto macrophages and minimize the number of dead tumor cells present in the coculture. One possible solution is to stain the tumor cells with a viability dye prior to adding them to macrophages, but we have found this to compromise viability of the tumor cells, and it does not account for cell death that occurs after the start of coculture. Finally, the ratio of tumor cells to macrophages impacts the phagocytosis rate. If the tumor to macrophage ratio is low in some conditions, this might artificially inflate the measured phagocytosis rate. As such, it is important to keep the tumor cell to macrophage ratio consistent between conditions.

TROUBLESHOOTING Problem 1
Incomplete collection of cells from the 12-well plates (related to step 13b).

Potential solution
Macrophages adhere strongly to tissue culture-treated plates. Allow the plates to incubate with trypsin for 10-15 min at 37 C and pipet vigorously at points all around the circumference of the well to lift cells. If there are still cells attached, repeat the pipetting process with 1 mL of HBSS in each well to collect the remaining cells. We do not recommend using a cell scraper as this can damage the macrophages. Longer incubations up to 30 min with trypsin diluted in HBSS (no calcium or magnesium and 0.025% trypsin) or temperature-sensitive plates may be used to facilitate macrophage detachment.

Problem 2
High measured phagocytosis rate across all conditions (related to step 20).

Potential solution
It is critical to maintain the health of the tumor cells that are added to the macrophages. Unhealthy or dead tumor cells will lead to high rates of efferocytosis, making all CD45 + cells also positive for the tumor cell marker. Keep the tumor cells on ice throughout handling and optimize the staining procedure to minimize toxicity. Ensure that tumor cells are thoroughly washed before plating onto macrophages so that no residual CFSE or other stain is carried to the coculture.

Problem 3
No phagocytosis is observed (related to step 20).

Potential solution
This could be dependent on the cell line, as we have found that phagocytosis rates differ among different tumor cell lines. The baseline rate of phagocytosis of live tumor cells by macrophages should be low. 4,5 Inclusion of LCL-161 or other positive control is critical to verify the technical success of the procedure. Tumor-specific antibodies that engage Fc receptors on macrophages should also induce phagocytosis, 6,7 particularly when combined with CD47 blocking antibodies or the use of CD47À/À tumor cell lines. [8][9][10][11] RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Li Qiang (Li_Qiang@dfci.harvard.edu). Materials availability This cell line 6694c2-zsGreen-NLS is available upon request.

Data and code availability
This study did not generate any datasets or code.